Topoisomerases are essential enzymes that act to relieve topological problems with DNA. They bind to DNA and carry out a coordinated set of actions to alter the topology of DNA. This proposal focuses on studying DNA gyrase, a bacterial paralog to human topoisomerase II and a paradigm for all type IIA enzymes. DNA gyrase uses a two-gate strand passage mechanism to change the topological state of DNA. This mechanism includes cleavage of one DNA strand, called the G-segment, and passage of a second strand, called the T-segment, through the cut G-segment. While type II topoisomerases have been studied extensively there are still unresolved details about T-segment passage through the enzyme. Here I propose both structural and biochemical experiments to better understand T-segment passage and overall enzyme architecture. Objective/Hypothesis The underlying hypothesis of the proposed work is that T-segment passage is coordinated by interactions with different domains as it travels through the enzyme. To test this hypothesis, the objective of this study is to provide structural details of the DNA gyrase complex with the G-segment and T-segment bound. This study aims to gain mechanistic information about T-segment passage through the C-gate of the enzyme using biochemical studies. These studies will provide information on the movement of the T-segment during the catalytic cycle.Specific Aims (1) Determine the mechanistic underpinnings of T-segment passage from the N-gate through the DNA gate. (2) Elucidate the molecular interactions that mediate T-segment passage through the C-gate.To better understand the passage of DNA through DNA gyrase I will use cryo-electron microscopy and biochemical experiments. To overcome hurdles that have prevented obtaining an atomic level structure of DNA gyrase in past studies, I plan to use a unique circular DNA to bind to DNA gyrase. My preliminary studies suggest that the use of this DNA not only supports DNA gyrase binding, but also helps restrain flexible domains within DNA gyrase. I hypothesize that this unique DNA will allow for the structural characterization of the G-segment and T-segment bound in the enzyme simultaneously. I will also perform biochemical experiments to determine how residues that line the C-gate recognize and allow the T-segment to exit the enzyme. These studies will give new insights into the mechanism of type II topoisomerases.Topoisomerases are targets for drugs that have been used to treat a variety of malignancies, including bladder, lung, and stomach cancers. Many of these drugs work by stalling topoisomerases after DNA cleavage. A potential side effect of their use is the development of secondary malignancies and some cancers can become immune to these compounds. Gaining novel mechanistic information about type II topoisomerases will provide a tool to improve current anticancer drugs or find alternate ways to target this enzyme.
|Effective start/end date||9/1/20 → 10/22/21|
- American Cancer Society (PF-20-041-01 - DMC)
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